Method and apparatus for conducting an array of chemical reactions on a support surface

ABSTRACT

The invention provides apparatus and methods for making arrays of functionalized binding sites on a support surface. The invention further provides apparatus and methods for sequencing oligonucleotides and for identifying the amino acid sequence of peptides that bind to biologically active macromolecules, by specifically binding biologically active macromolecules to arrays of peptides or peptide mimetics.

This is a continuation of application Ser. No. 08/068,540, filed May 27,1993; now U.S. Pat. No. 5,474,796 which in turn is acontinuation-in-part of application Ser. No. 07/754,614, filed Sep. 4,1991 now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to methods for conducting a large number ofchemical reactions on a support surface, methods for making the supportsurface, and the support surface itself.

2. Summary of the Related Art

Proposals for the direct sequencing of DNA by hybridization with arraysof oligonucleotides are known in the art. Drmanac et al., Genomics 4;114 (1989) proposes hybridization array-mediated DNA sequencing bybinding target DNA to a dot blot membrane, followed by probing with anarray of oligonucleotides. Khrapko et al., FEBS Letters 256, 118 (1989)proposes hybridization array-mediated DNA sequencing by binding theoligonucleotide array to a support membrane, followed by probing withtarget DNA.

Synthesis of arrays of bound oligonucleotides or peptides is also knownin the art. Houghton, in the Multiple Peptide System product brochuredescribes the T-bag method, in which an array of beads is physicallysorted after each interaction. This method becomes unwieldy for thepreparation of large arrays of oligonucleotides. Geysen et al., J.Immunol. Methods 102; 259 (1987) discloses the pin method for thepreparation of peptide arrays. The density of arrays that may beproduced by this method is limited, and the dipping procedure employedin the method is cumbersome in practice. Southern, Genome Mapping andSequencing Conference. May 1991, Cold Spring Harbor, N.Y., disclosed ascheme for oligonucleotide array synthesis in which selected areas on aglass plate are physically masked and the desired chemical reaction iscarried out on the unmasked portion of the plate. In this method it isnecessary to remove old mask and apply a new one after each interaction.Fodor et al., Science 251; 767 (1991) describes a method forsynthesizing very dense 50 micron arrays of peptides(and potentiallyoligonucleotides) using mask-directed photochemical deprotection ofsynthetic intermediates. This method is limited by the slow rate ofphotochemical deprotection and by the susceptibility to side reactions(e.g., thymidine dimer formation) in oligonucleotide synthesis. Khrapkoet al, FEBS Letters 256; 118 (1989) suggests simplified synthesis andimmobilization of multiple oligonucleotides by direct synthesis on a twodimensional support, using a printer-like device capable of samplingeach of the four nucleotides into given dots on the matrix. However, noparticulars about how to make or use such a device are provided.

Some methods for permanently attaching oligonucleotides to glass platesin a manner suitable for oligonucleotide synthesis are known in the art.Souther, Chem. abst. 113; 152979r (1990) describes a stable phosphateester linkage for permanent attachment of oligonucleotides to a glasssurface. Mandenius et al., Anal. Biochem. 157; 283 (1986) teaches thatthe hydroxyalkyl group resembles the 5'-hydroxyl of oligonucleotides andprovides a stable anchor on which to initiate solid phase synthesis.

The related art contains numerous ideas and information related toarrays of chemical reactants on a solid support. However, existing orsuggested methods are limited, and do not conveniently and reliablyproduce the very large, high density arrays. There is, therefore, a needfor new methods for preparing large high density arrays of reactivesites. Ideally, such methods should utilized relatively simple machineryto produce large, dense arrays of solid phase bound reactants in areproducible and rapid manner.

SUMMARY OF THE INVENTION

This invention provides a method for conducting a large number ofchemical reactions on a support surface. Solutions of chemical reactantsare added to functionalized binding sites on the support surface bymeans of a piezoelectric pump. This pump deposits microdroplets ofchemical reactant solution onto the binding sites. The chemical reactantat each binding site is separated from the others by surface tension.Typically, the support surface has 10-10⁴ functionalized binding sitesper cm² and each functionalized binding site is about 50-2000 microns indiameter. Typically, the amounts of reagents added to each binding siteis in a volume of about 50 picoliter to 2 microliter. The reactions atthe functionalized binding site may form covalent bonds such as estersor amide bonds or may involve non-covalent specific binding reactionssuch as antibody/antigen binding or oligonucleotide specific binding.The invention also includes array plates and methods for making thearray plates.

Typically, the array plates are made by the process set out in FIG. 2Aby

(a) coating a support surface with a positive or negative photoresistsubstance which is subsequently exposed and developed to create apatterned region of a first exposed support surface;

(b) reacting the first support surface with a fluoroalkylsilane to forma stable fluoroalkylsiloxane hydrophobic matrix on the first supportsurface;

(c) removing the remaining photoresist to expose a second supportsurface; and

(d) reacting the second support with a hydroxy or aminoalkylsilane toform derivatized hydrophilic binding site regions.

The preferred siloxane reaction product of the present invention istridecafluoro-1,1,2,2-tetrahydrooctyl siloxane. In FIG. 2A, the hatchedlines are the solid support, "S1" represents a first exposed supportsurface site, "S1-F" is a hydrophobic fluoroalkylsilane site, and"S1-OH" is a derivatized hydrophilic binding site.

Alternatively, the array plates can be made by the process set out inFIG. 2B by

(a) reacting a support surface with a hydroxy or aminoalkylsilane toform a derivatized hydrophilic support surface;

(b) reacting the support surface form step (a) with o-nitrobenzylcarbonyl chloride as a temporary photolabile blocking to provide aphotoblocked support surface;

(c) exposing the photoblocked support surface of step (b) to lightthrough a mask to create unblocked areas on the support surface withunblocked hydroxy or aminoalkylsilane;

(d) reacting the exposed surface of step (c) with perfluoroalkanoylhalide or perfluoroalkylsulfonyl halide to form a stable hydrophobic(perfluoroacyl or perfluoroalkylsulfonamido) alkyl siloxane matrix; and

(e) exposing this remaining photoblocked support surface to createpatterned regions of the unblocked hydroxy- or aminoalkylsilane to formthe derivatized hydrophilic binding site regions.

The preferred siloxanes of the present invention are3-perfluorooctanoyloxy propylsiloxane and 3-perfluorooctanesulfonamidopropylsiloxane. In FIG. 2B, the hatched lines are the solid support,"-A" represents a hydrophilic support site, "-A B" represents atemporary photolabile blocked support site, and "-A F" represents ahydrophobic site.

The invention also provides a method for determining or confirming thenucleotide sequence of a target nucleic acid. The target nucleic acid islabelled by conventional methods and hybridized to an oligonucleotidesof known sequence previously bound to sites on the array plate. Thearray plate having bound labelled target nucleic acid is then washed atappropriate stringency and the presence and location of bound labelledtarget nucleic acid is determined using scanning analyzers. Since thesequence of the covalently attached oligonucleotide in each element onthe array is known, this allows the unambiguous determination of thenucleotide sequence of the target nucleic acid.

The methods of the invention may also be applied to the determination ofpeptides or peptide mimetics that bind biologically active receptors. Inthis aspect, peptide arrays of known sequence can be applied to glassplates using the same piezoelectric pump/surface tension wall methoddescribed supra. The resulting array of peptides can then be used inbinding analyses with biologically active receptor ligands to screen forpeptide mimetics of receptor agonists and antagonists. Thus, theinvention provides a method for producing peptide array plates, peptidearray plates having covalently bound peptides separated by surfacetension areas, and methods of using such peptide array plates to screenfor peptide mimetics of receptor agonists and antagonists.

Those skilled in this art will recognize a wide variety of binding siteand chemical reactants for forming either covalent bonds or for specificbinding reagents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Hybridization analysis using arrays of trimers. Individual dotsthat have bound the DNA fragment are underlined.

FIG. 2A: Illustrates the formation of an array surface that is ready forsolid phase synthesis.

FIG. 2B: Illustrates O-Nitrocarbamate array making chemistry.

FIG. 3: Surface tension wall effect at the dot-interstice interface. Thedroplet containing solid phase synthesis reagents does not spread beyondthe perimeter of the dot due to the surface tension wall.

FIG. 4: Hydrogen-phosphonate solid phase oligonucleotide synthesis on anarray surface prepared according to Example 1.

FIG. 5: Top and side views of a piezoelectric impulse jet of the typeused to deliver solid phase synthesis reagents to individual dots in thearray plate synthesis methods according to the invention.

FIG. 6: Use of a piezoelectric impulse jet head to deliver blockednucleotides and activating agents to individual dots on an array plate.The configuration shown has a stationary head/moving plate assembly.

FIG. 7: Enclosure for array reactions showing array plate, sliding coverand manifolds for reagent inlet and outlet.

DETAILED DESCRIPTION OF THE INVENTION

The practice of present invention can include a number of photoresistsubstances. These substances are readily known to those of skill in theart. For example, an optical positive photoresist substance (e.g., AZ1350 (Novolac™ type-Hoechst Celanese™) (Novolac™ is a proprietarynovolak resin, which is the reaction product of phenols withformaldehyde in an acid condensation medium)) or an E-beam positivephotoresist substance (e.g., EB-9 (polymethacrylate by Hoya™)) can beused.

A number of siloxane functionalizing reagents can be used, for example:

1. Hydroxyalkyl siloxanes

(Silylate surface, functionalize with diborane, and H₂ 0₂ to oxidize thealcohol)

a. allyl trichlorochlorosilane→→3-hydroxypropyl

b. 7-oct-1-enyl trichlorochlorosilane→→8-hydroxyoctyl

2. Diol (dihydroxyalkyl) siloxanes

(silylate surface, and hydrolyze to diol)

a. glycidyl trimethoxysilane→→(2,3-dihydroxypropyloxy)propyl

3. Aminoalkyl siloxanes (amines require no intermediate functionalizingstep)

a. 3-aminopropyl trimethoxysilane→3-aminopropyl

4. Dimeric secondary aminoalkyl siloxanes

a. bis (3-trimethoxysilylpropyl) amine→bis (silyloxylpropyl) amine

In addition, a number of alternative functionalized surfaces can be usedin the present invention. These include the following:

1. Polyethylene/polypropylene functionalized by gamma irradiation orchromic acid oxidation, and reduction to hydroxyalkyl surface.

2. Highly crosslinked polystyrene-divinylbenzene derivatized bychloromethylation, and aminated to benzylamine functional surface.

3. Nylon--the terminal aminohexyl groups are directly reactive.

4. Etched, reduced polytetrafluoroethylene.

There are two important characteristics of the masked surfaces inpatterned oligonucleotide synthesis. First, the masked surface must beinert to the conditions of ordinary oligonucleotide synthesis; the solidsurface must present no free hydroxy, amino or carboxyl groups to thebulk solvent interface. Second, the surface must be poorly wet by commonorganic solvents such as acetonitrile and the glycol ethers, relative tothe more polar fuctionalized binding sites.

The wetting phenomenon is a measure of the surface tension or attractiveforces between molecules at a solid-liquid interface, and is defined indynes/cm². Fluorocarbons have very low surface tension because of theunique polarity (electronegativity) of the carbon-flourine bond. Intightly structured Langmuir-Blodgett type films, surface tension of alayer is primarily determined by the percent of fluorine in the terminusof the alkyl chains. For tightly ordered films, a single terminaltrifluoromethyl group will render a surface nearly as lipophobic as aperfluoroalkyl layer. When fluorocarbons are covalently attached to anunderlying derivatized solid (highly crosslinked polymeric) support, thedensity of reactive sites will generally be lower than Langmuir-Blodgettand group density. However, the use of perfluoroalkyl masking agentspreserves a relatively high fluorine content in the solvent accessibleregion of the supporting surface.

There are also two important characteristics of the derivatized regionsin patterned oligonucleotide synthesis. The surface must be compatiblewith the method of detection of hybridization. Radioactivity is largelybeing replaced by spectroscopic, chemiluminescent and fluorescentdetection techniques in DNA research. It is desirable that the surfacebe optically transparent. A second important characteristic is that thelinkage of the penultimate oligonucleotide to the surface have highchemical stability, at least equal to that of the polyphosphate backbonein DNA.

The optical properties of glass (polytetrasiloxane) are unsurpassed fordetection purposes. Further, there are numerous techniques developed bythe semiconductor industry using thick films (1-5 microns) ofphotoresists to generate masked patterns of exposed glass surfaces. Thebest method to derivatize the first exposed glass surface is withvolatile fluoroalkyl silanes using gas phase diffusion to create closelypacked lipophobic monolayers. The polymerized photoresist provides aneffectively impermeable barrier to the gaseous fluoroalkyl silane duringthe time period of derivatization of the exposed region. Followinglipophobic derivatization however, the remaining photoresist can bereadily removed by dissolution in warm organic solvents (methyl,isobutyl, ketone, or N-methyl pyrrolidone) to expose a second surface ofraw glass, while leaving the first applied silane layer intact. Thissecond region glass can then be derivatized by either solution or gasphase methods with a second, polar silane which contains either ahydroxyl or amino group suitable for anchoring solid phaseoligonucleotide synthesis.

Siloxanes have somewhat limited stability under strongly alkalineconditions. Conditions such as 0.1 N sodium hydroxide, typicallyemployed to strip probes from nylon hybridization membranes, should beavoided for reusable glass based hybridization arrays.

Teflon (polytetrafluoroethylene) itself would provide an ideallipophobic surface. Patterned derivatization of this type of materialcan be accomplished by reactive ion or plasma etching through a physicalmask or using an electron beam, followed by reduction to surfacehydroxymethyl groups. However, the opacity of teflon at visiblewavelengths severely restrict the applicable methods for detection ofhybridization.

Depending on the ultimate application, other organic polymers havedesirable characteristics for patterned oligonucleotide synthesis.Polypropylene is relatively transparent to visible light. It can besurface derivatized by chromic acid oxidation, and converted to hydroxy-or aminomethylated surfaces which provide oligonucleotide synthesisanchors of high chemical stability. Highly crosslinkedpolystryene-divinylbenzene (ca. 50%) is non-swellable, and can bereadily surface derivatized by chloromethlylation and subsequentfunctional group manipulation. Nylon provides an initial surface ofhexylamino groups.

The lipophobic patterning of these surfaces can be effected using thesame type of solution based thin film masking techniques and gas phasederivatization as glass, or by direct photochemical patterning usingo-nitrobenzylcarbonyl blocking groups. Perfluoroalkyl carboxylic andsulfonic acid derivatives rather than silanes are now used to providethe lipophobic mask of the underlying surface during oligonucleotidesynthesis.

The solution of chemical reactant can be added to the functionalizedbinding site through utilization of a piezoelectric pump (FIG. 5) in anamount where the solution of chemical reactant at each binding site isseparate from the solution of chemical reactant at other binding sitesby surface tension. As described more fully infra, in the pump depictedin FIG. 5, reactant solution is inserted through the inlet (2) into thechamber (6) formed between the upper (1) and lower (5) plates of thepiezo. Application of a voltage difference across the upper and lowerplates causes compression of the piezo, forcing a microdroplet (4) outthrough the nozzle (3).

FIG. 3 depicts the deposition of the reactant solution on afunctionalized binding site and subsequent reaction with the surface. Amicro-droplet of solution (FIG. 3(a)) is deposited on the functionalizedbinding site (center cross-hatched region in FIG. 3(b)). Because of thedifferences in wetting properties of the reactant solution on thefunctionalized binding site and the surrounding surface, themicro-droplet of the reactant solution beads on the functionalizedbinding site and the reactants in solution react with the surface (FIG.3(c)).

The piezoelectric pump that may be utilized in the invention deliversminute droplets of liquid to a surface in a very precise manner. Thepump design is similar to the pumps used in ink jet printing. Thepicopump is capable of producing 50 micron or 65 picoliter droplets atup to 3000 Hz and can accurately hit a 250 micron target in a 900° C.oven at a distance of 2 cm in a draft free environment. Preferredembodiments of the apparatus according to the invention are set forth inExample 3.

Alternative pump designs should take into account the following physicaland mechanical considerations for reliable performance to be obtained.When a non-compressible fluid inside of a pumping cavity is subjected toa rapid strong pressure pulse, the direction of flow of the liquid fromthe cavity is determined primarily by the inertial resistance of theliquid placed. There is more liquid, and thus resistance to flow, on theinlet side than through the nozzle port. The column of liquid that isforced out of the nozzle begins to neck off as a result of surfacetension. The stream breaks as the piezoelectric is de-energized, withthe remaining column of liquid drawn back into the nozzle. The dropletthat has necked off continues its flight with the velocity it achievedin the initial acceleration. Typically, the ejection velocity is about1-2 meters/sec.

In normal printing applications using 150 micron drops of viscouswater-based inks, the head speed is typically about 0.5 meter/sec. Thismotion adds a transverse velocity component to the droplet trajectoryand can affect aiming accuracy. It may also cause the drop to skip whenit hits a surface. Droplets fired from a stationary head tend toevaporate more slowly because they follow in the vapor trail of thepreceding drop. The heads work most reliably when the inlet supply linesare not required to flex and the liquids are not subjected toacceleration forces.

The size of the drop is determined primarily by the surface tension ofthe solution and by the diameter of the pump nozzle. The smaller thedroplet, the faster it will evaporate and the more its trajectory willbe affected by drafts. Nozzles smaller than 25 microns tend to becomeplugged with dust particles. For water, the drop diameter isapproximately 1.5 times the nozzle diameter. Typically, drops will notvary in size by more than 5%. We have shown that the jet will alsosuccessfully eject a variety of polar solvents, including CH₃ CN andMeOH. With these less viscous solvents, too forceful an ejection pulsemay result in the formation of a series of trailing satellite dropletsin addition to the primary drop. The duration of the pulse also affectsatelliting.

After the cavity has returned to its original state, a period of timemust be allowed for the nozzle to refill by capillary action beforeanother cycle of pulsing can be initiated. It is important for thenozzle refill only to the top of the orifice, but the liquid meniscusnot spread out onto the front face of the jet. This is prevented bysilanizing the face to reduce its surface tension. The head is alsooperated under slight negative pressure to prevent overfilling. The aimof the drop is in the axial direction of the nozzle, but defects in theface coating can affect the trajectory.

Arrays of nozzles with up to 64 independent pumping chambers but acommon inlet supply have been fabricated. It is important that eachchamber inlet have some restriction so that operation of one pumpingchamber does not affect the others. The separation between nozzles istypically 400 microns for printing applications, but denser arrays canbe produced either by interleaving the transverse motion of the targetor decreasing the nozzle spacing.

EXAMPLE 1 Preparation of Array Plates Ready for Oligonucleotide orPeptide Assembly

The hybridization array is synthesized on a glass plate. The plate isfirst coated with the stable fluorosiloxane3-(1,1-dihydroperfluoroctyloxy) propyltriethoxysilane. A CO₂ laser isused to ablate off regions of the fluorosiloxane and expose theunderlying silicon dioxide glass. The plate is then coated withglycidyloxypropyl trimethoxysilane, which reacts only on the exposedregions of the glass to form a glycidyl epoxide. The plate is nexttreated with hexaethyleneglycol and sulfuric acid to convert theglycidyl epoxide into a hydroxyalkyl group, which acts as a linker arm.The hydroxyalkyl group resembles the 5'-hydroxide of nucleotides andprovides a stable anchor on which to initiate solid phase synthesis. Thehydroxyalkyl linker arm provides an average distance of 3-4 nm betweenthe oligonucleotide and the glass surface. The siloxane linkage to theglass is completely stable to all acidic and basic deblocking conditionstypically used in oligonucleotide or peptide synthesis. This scheme forpreparing array plates is illustrated in FIGS. 2(A) and 2(B) and waspreviously discussed.

EXAMPLE 2 Assembly of Oligonucleotides on the Array Plates

The hydroxyalkylsiloxane surface in the dots has a surface tension ofapproximately γ=47, whereas the fluoroxysilane has a surface tension ofγ=18. For oligonucleotide assembly, the solvents of choice areacetonitrile, which has a surface tension of γ=29, and diethylglycoldimethyl ether. The hydroxyalkylsiloxane surface is thus completely wetby acetonitrile, while the fluorosiloxane masked surface between thedots is very poorly wet by acetonitrile. Droplets of oligonucleotidesynthesis reagents in acetonitrile are applied to the dot surfaces andtend to bead up, as shown in FIG. 3. Mixing between adjacent dots isprevented by the very hydrophobic barrier of the mask. The contact anglefor acetonitrile at the mask-dot interface is approximately θ=43°. Theplate effectively acts as an array microliter dish, wherein theindividual wells are defined by surface tension rather than gravity. Thevolume of a 40 micron droplet is 33 picoliter. The maximum volumeretained by a 50 micron dot is approximately 100 picoliter, or about 3droplets. A 100 micron dot retains approximately 400 picoliter, or about12 droplets. At maximum loading, 50 micron and 100 micron dots bindabout 0.07 and 0.27 femtomoles oligonucleotide, respectively.

Assembly of oligonucleotides on the prepared dots (FIG. 2B, bottom) iscarried out according to the H-phosphonate procedure (FIG. 4), or by thephosphoroamidite method. Both methods are well known to those ofordinary skill in the art. Oligonucleotide and Analogs, A PracticalApproach (F. Eckstein ed., 1991). Delivery of the appropriate blockednucleotides and activating agents in acetonitrile is directed toindividual dots using the picopump apparatus described in Example 3. Allother steps, (e.g., DMT deblocking, washing) are performed on the arrayin a batch process by flooding the surface with the appropriatereagents. An eight nozzle piezoelectric pump head is used to deliver theblocked nucleotides and activating reagents to the individual dots, anddelivering droplets at 1000 Hz, requires only 32 seconds to lay down a512×512 (262 k) array. Since none of the coupling steps have criticaltime requirements, the difference in reaction time between the first andlast droplet applied is insignificant.

EXAMPLE 3 Construction of Piezoelectric Impulse Jet Pump Apparatus

Piezoelectric impulse jets are fabricated from Photoceram (CorningGlass, Corning, N.Y.), a UV sensitive ceramic, using standardphotolithographic techniques to produce the pump details. The ceramic isfired to convert it to a glassy state. The resulting blank is thenetched by hydrogen fluoride, which acts faster in exposed then innonexposed areas. After the cavity and nozzle details are lapped to theappropriate thickness in one plate, the completed chamber is formed bydiffusion bonding a second (top) plate to the first plate. The nozzleface is lapped flat and surface treated, then the piezoelectric elementis epoxied to the outside of the pumping chamber. When the piezoelectricelement is energized it deforms the cavity much like a one-sidedbellows, as shown in FIG. 5.

To determine the appropriate orifice size for accurate firing ofacetonitrile droplets, a jet head with a series of decreasing orificesizes is prepared and tested. A 40 micron nozzle produces droplets ofabout 65 picoliter.

A separate nozzle array head is provided for each of the fournucleotides and a fifth head is provided to deliver the activatingreagent for coupling. The five heads are stacked together with amechanically defined spacing. Each head has an array of eight nozzleswith a separation of 400 microns.

The completed pump unit is assembled with the heads held stationary andthe droplets fired downward at a moving array plate as shown in FIG. 6.The completed pump unit assembly (3) consists of nozzle array heads(4-7) for each of the four nucleotidase and a fifth head (8) foractivating reagent. When energized, a microdroplet (9) is ejected fromthe pump nozzle and deposited on the array plate (1) at a functionalizedbinding site (2).

A plate holding the target array is held in a mechanical stage and isindexed in the X and Y planes beneath the heads by a synchronous screwdrives. The mechanical stage is similar to those used in small millingmachines, microscopes and microtomes, and provides reproduciblepositioning accuracy better than 2.5 microns or 0.1 mil. As shown inFIG. 7, the plate holder (3) is fitted with a slotted spacer (4) whichpermits a cover plate (5) to be slid over the array (6) to form anenclosed chamber. Peripheral inlet (1) and outlet (2) ports are providedto allow the plate to be flooded for washing, application of reagentsfor a common array reaction, or blowing the plate dry for the next dotarray application cycle.

Both the stage and head assembly are enclosed in a glove box which canbe evacuated or purged with argon to maintain anhydrous conditions. Withthe plate holder slid out of the way, the inlet lines to the heads canbe pressurized for positive displacement priming of the head chambers orflushing with clean solvent. During operation, the reagent vials aremaintained at the ambient pressure of the box.

With a six minute chemistry cycle time, the apparatus can produce 10-merarray plates at the rate of 1 plate or 106 oligonucleotides per hour.

EXAMPLE 4 Use of Oligonucleotide Array Plates to Determine theNucleotide Sequence of a Target Nucleic Acid

The oligonucleotide array plate is prepared as described in Examples 1and 2, using the apparatus described in Example 3. The array containsoligonucleotides having 10 nucleotides each (10-mers). The synthesis iscarried out such that each oligonucleotide element, moving in a 5'-3'direction, is identical to the preceding element in nucleotide sequence,except that it deletes the 5'-most nucleotide, and adds a new 3'-mostoligonucleotide. In this way the total array represents every possiblepermutation of the 10-mer oligonucleotide. Oligonucleotides are spacedat 7 nm intervals to provide an oligonucleotide loading density of3.4×10⁻⁻¹² moles/cm², or 2.6×10⁻¹⁶ moles per 100 micron element. Thetarget nucleic acid is used to probe the oligonucleotide array plate.The probe is labelled with 1000 Ci/nmol p³². The labelled probe iscontacted with the oligonucleotide array plate for hybridization in a 10nM solution of probe in 3M Me₄ NCl at 42° C. At 10% hybridization andwash efficiency, each oligonucleotide element dot having an exact matchwith the probe binds 26 attomoles of probe. Radiolabel binding isdetected using a Bio-Image Analyzer™ (Fuji, Waltham, Mass.). The patternof binding is assessed and the nucleotide sequence of the probe nucleicacid is determined by ordering the nucleotide sequence according to theknown sequences of the oligonucleotide elements, as shown in FIG. 1.

FIG. 1 depicts a sequencing arrangement based on a matrix of trimeroligonucleotides bound to the array plate. FIG. 1(a) is the basic matrixconsisting of the four nucleotides. FIG. 1(b) is the complete trimermatrix, representing each of the 43 trimer permutations. The underlinedelements in the array represent sites to which the target nucleic acidis bound. FIG. 1(c) depicts how a sequence complementary to the targetnucleic acid is constructed from the known sequences of the sites towhich the target nucleic acid is bound.

What is claimed is:
 1. An array plate comprising a support surfacecomprising a covalently linked layer of inert siloxane, wherein saidcovalently linked layer defines an array of 10 to 10⁴ sites per cm²,which do not have said covalently linked layer, and which are about50-2000 microns in diameter, and wherein chemical reactant solutionslocalize to said sites via surface tension.
 2. The array plate accordingto claim 1, wherein said siloxane istridecafluro-1,1,2,2-tetrahydrooctyl siloxane.
 3. The array plateaccording to claim 1, wherein said sites are functionalized to bind orcovalently link a nucleic acid.
 4. The array plate according to claim 3,wherein said sites comprise siloxane compounds selected from the groupconsisting of hydroxyalkyl siloxane, dihydroxyalkyl siloxanes, andaminoalkyl siloxanes.
 5. A method of using an array plate comprising asupport surface comprising a covalently linked layer of inert siloxane,wherein said covalently linked layer defines an array of 10 to 10⁴ sitesper cm², which do not have said covalently linked layer, and whereinchemical reactant solutions localize to said sites via surface tension,said method comprising depositing a solution of nucleic acid reagent onsaid array plate in an amount such that said solution of nucleic acidreagent at said sites stays separated from said solution of nucleic acidreagent at other sites due to surface tension.
 6. A method of using anarray plate for conducting nucleic acid reactions, wherein said arrayplate comprises a support surface comprising a covalently linked layerof inert siloxane, said covalently linked layer defines an array of 10to 10⁴ functionalized binding sites per cm², which do not have saidcovalently linked layer, such that chemical reactant solutions localizeto said sites via surface tension, said method comprising depositing asolution of nucleic acid reagent on said array plate to chemically reactwith said functionalized binding sites in an amount such that saidsolution of nucleic acid reagent at each said binding site staysseparated from said solution of nucleic acid reagent at other sites dueto surface tension.
 7. The method according to claim 5 or 6, whereinsaid depositing is performed using an ink jet printing apparatus.
 8. Themethod according to claim 5 or 6, wherein said depositing is performedusing a piezoelectric pump.
 9. The method according to claim 5 or 6,wherein said amount is about 50 picoliters to about 2 microliters.
 10. Amethod according to claim 6, wherein said nucleic acid reactions formcovalent bonds between said nucleic acid reagent and said functionalizedbinding sites.
 11. A method of using an array plate for conductingnucleic acid reactions, wherein said array plate comprises a supportsurface comprising a covalently linked layer of inert siloxane, saidcovalently linked layer defines an array of 10 to 10⁴ functionalizedbinding sites per cm², which do not have said covalently linked layer,and said functionalized binding sites are more polar than thesurrounding surface, said method comprising depositing a solution ofnucleic acid reagent on said functionalized binding sites in an amountsuch that said solution of nucleic acid reagent at each of said bindingsites stays separated from said solution of nucleic acid reagent atother sites due to differential wetting of said more polarfunctionalized binding sites.
 12. The method according to claim 11,wherein said depositing is performed using an ink jet printer apparatus.13. The method according to claim 11, wherein said depositing isperformed using a piezoelectric pump.
 14. The method according to claim11, wherein said amount is about 50 picoliters to about 2 microliters.15. A method according to claim 11, wherein said nucleic acid reactionforms covalent bonds between said nucleic acid reagent and saidfunctionalized binding site.